Post-natal transplantation of factor viii-expressing cells for treatment of hemophilia

ABSTRACT

Disclosed herein are method of treating hemophilia A in a subject comprising injecting the subject with mesenchymal stromal/stem cells (MSC) modified to express high levels of Factor VIII protein. The MSC are isolated prenatally, at birth, or after the subject&#39;s birth. The modified MSC may also express high levels von Willebrand factor protein.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 62/549,280, filed Aug. 23, 2017, the contents of this application isherein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant number1R01HL130856-01A1 awarded by the U.S. National Institutes of Health(NIH). The government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file namedSEQ_WFIRM17-908.txt, created on Aug. 23, 2018, and having a size of 176KB and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND

Factor VIII is an essential blood clotting factor. The proteincirculates in the bloodstream in an inactive form, bound to anothermolecule called von Willebrand factor, until an injury that damagesblood vessels occurs. In response to injury, coagulation factor VIII isactivated and separates from von Willebrand factor. The active proteininteracts with another coagulation factor called Factor IX. Thisinteraction sets off a chain of additional chemical reactions that forma blood clot.

Hemophilia A (HA) is the most common inheritable coagulation deficiency,affecting 1 in 5000 boys, approximately 60% of whom present with thesevere form of the disease. Mutations in the Factor VIII gene thatresult in decreased or defective Factor VIII protein give rise to HA, arecessive X-linked disorder. Individuals with severe HA experiencerecurrent hematomas of subcutaneous connective tissue/muscle, internalbleeding, and frequent hemarthrosis, leading to chronic debilitatingarthropathies. Current treatment is frequent infusions of Factor VIII(plasma-derived or recombinant) to maintain hemostasis, which greatlyimproves quality of life for many HA patients. While current therapeuticproducts for HA offer reliable prophylactic and therapeutic efficacy,they are very expensive and do not cure the underlying disease, thusrequiring administration for the entire life of the patient. Inaddition, more than 30% of patients with severe HA develop inhibitoryantibodies to the infused Factor VIII therapeutic, placing them indanger of treatment failure. This is a significant and seriouscomplication/challenge in the clinical management/treatment of HA. Whileprotein-based immune tolerance induction (ITI) therapy has been usedwith some success in this patient group, its cost extends into themillions of dollars per patient, it is only effective in about 60% ofpatients, and its mechanism of action is largely unknown. Theseshortcomings with existing therapy for patients who develop inhibitorshighlight the need for innovative approaches to surmount thisimmunological hurdle.

BRIEF SUMMARY

In one aspect, provided are methods of treating a subject diagnosed withhemophilia A, the method involving the steps of (a) modifyingmesenchymal stem/stromal cells (MSC) to express high levels of FactorVIII protein thereby generating modified MSC, the MSC comprisingbone-marrow MSC isolated from the subject; (b) generating an expandedmodified MSC population by in vitro culturing the modified MSC; and (c)injecting MSC from the expanded modified MSC population into thesubject.

In another aspect, provided are methods of treating a subject prenatallydiagnosed as having hemophilia A, the method involving the steps of (a)modifying mesenchymal stem/stromal cells (MSC) to express high levels ofFactor VIII protein thereby generating modified MSC, the MSC comprisingMSC isolated from at least one of amniotic fluid, placental tissue, orumbilical cord tissue obtained at the time of the subject's birth orprenatally from the subject's mother; (b) generating an expandedmodified MSC population by in vitro culturing the modified MSC; and (c)injecting MSC from the expanded modified MSC population into thesubject.

The above described and many other features and attendant advantages ofembodiments of the present disclosure will become apparent and furtherunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

These figures are intended to be illustrative, not limiting. Althoughthe aspects of the disclosure are generally described in the context ofthese figures, it should be understood that it is not intended to limitthe scope of the disclosure to these particular aspects.

FIG. 1 shows the complete absence of any FVIII antigen/cross-reactivematerial (CRM) in the hemophilia A sheep according to some aspects ofthe disclosure.

FIG. 2 shows assessment of phenotypic markers in MSC isolated from sheepaccording to some aspects of the disclosure.

FIG. 3 shows a schematic diagram of an ovine Factor VIII transgeneexpression vector according to some aspects of the disclosure.

FIG. 4 shows a schematic diagram of an ovine vWF transgene expressionvector according to some aspects of the disclosure.

FIG. 5 shows endogenous expression of Factor VIII in MSC isolated fromamniotic fluid according to some aspects of the disclosure.

FIG. 6 shows endogenous expression of vWF in MSC isolated from amnioticfluid according to some aspects of the disclosure.

FIG. 7 shows exogenous expression of Factor VIII in MSC isolated fromamniotic fluid and transduced with a lentivector encoding Factor VIIIaccording to some aspects of the disclosure.

FIG. 8A shows assessment of phenotypic markers in PLCs according to someaspects of the disclosure.

FIG. 8B shows flow cytometric analysis of PLC constitutively expressedlevels of FVIII protein according to some aspects of the disclosure.

FIG. 8C shows assessment of normalized levels of PLC constitutivelyexpressed levels of FVIII protein according to some aspects of thedisclosure.

FIG. 9A shows assessment of normalized levels of secretion of FVIIIprotein by PLC engineered/transduced to express high levels of FVIIIaccording to some aspects of the disclosure.

FIG. 9B shows assessment of secretion of FVIII protein by transduced ascompared to non-transduced PLCs according to some aspects of thedisclosure.

FIGS. 10A-10C show assessment of phenotypic markers in transduced ascompared to non-transduced PLCs according to some aspects of thedisclosure.

FIG. 11A shows assessment of expression of Toll-like receptors (TLRs) inmcoET3-transduced PLCs as compared to non-transduced PLCs according tosome aspects of the disclosure.

FIG. 11B shows assessment of expression of stress molecules inmcoET3-transduced PLCs as compared to non-transduced PLCs according tosome aspects of the disclosure.

FIG. 12 shows assessment of normalized levels of PLC secretion of FVIIIprotein by non-transduced-PLC, lcoHSQ-PLC, lcoET3-PLC, ET3-PLC, andmcoET3-PLC according to some aspects of the disclosure.

DETAILED DESCRIPTION

Provided in this disclosure are methods of treatment for subjects havinghemophilia A. The methods are post-natal therapies comprisingadministering to a subject with hemophilia A autologous mesenchymalstem/stromal cells (MSC) that have been modified to express Factor VIII.Provided methods are effective as first-line therapies for subjects thathave been diagnosed prenatally or at an early age and who have notreceived Factor VIII therapy. Provided methods are also effective assecond-line therapies for the treatment of subjects that have beenreceiving Factor VIII therapy and, in some instances, have developed animmune response to standard infusion therapy of exogenous Factor VIII.The MSC used in the methods are isolated from biological samplesobtained prenatally or after the subject's birth. The MSC are modifiedto express high levels of Factor VIII protein. In some instances, theMSC are modified to express high levels of Factor VIII and high levelsof another protein, such as von Willebrand factor. The MSC may bemodified by the introduction of a transgene (for example, using a viralvector) or via genome-editing (for example, using the CRISPR/Cas9system). Administering the modified MSC to the subject results inengraftment of the modified cells. The engrafted cells produce FactorVIII on a continuing basis in the subject and provide long-lasting(ideally lifelong) therapeutic benefit to the subject by promoting bloodcoagulation.

In one aspect, provided is a method of treating a subject diagnosedclinically or genetically with hemophilia A comprising: (a) modifyingmesenchymal stem/stromal cells (MSC) to express high levels of FactorVIII protein thereby generating modified MSC, the MSC comprising bonemarrow MSC isolated from the subject; (b) generating an expandedmodified MSC population by in vitro culturing the modified MSC; and (c)injecting MSC from the expanded modified MSC population into thesubject. The bone marrow MSC express at least one of Stro-1 or CD146. Insome instances, the bone marrow MSC may be isolated based on expressionof at least one of Stro-1 or CD146.

In another aspect, provided is a method of treating a subject prenatallydiagnosed as having hemophilia A comprising: (a) modifying mesenchymalstem/stromal cells (MSC) to express high levels of Factor VIII proteinthereby generating modified MSC, the MSC comprising MSC isolated from atleast one of amniotic fluid, placental tissue, or umbilical cord tissueobtained at the time of the subject's birth or prenatally; (b)generating an expanded modified MSC population by in vitro culturing themodified MSC; and (c) injecting MSC from the expanded modified MSCpopulation into the subject. In some instances, the MSC are amnioticfluid MSC. In some instances, the MSC are placental MSC (PLC). In someinstances, the MSC are umbilical cord tissue MSC.

As used herein the terms treatment, treat, or treating refer to a methodof reducing one or more symptoms of a disease or condition. In someinstances, treatment results in a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100% reduction in the severity of one or more symptoms ofthe disease or condition. In some instances, treatment results in atleast a 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction in theseverity of one or more symptoms of the disease or condition. In someinstances, treatment results in a 100% reduction in the severity of oneor more symptoms of the disease or condition. For example, a method fortreating a disease is considered to be a treatment if there is a 5%reduction in one or more symptoms or signs. As used herein, controlrefers to the untreated condition. In some instances, the reduction canbe a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or anypercent reduction in between 10% and 100% as compared to native orcontrol levels. In some instances, the reduction can be at least a 65%,70%, 75%, 80%, 85%, 90%, or 95% reduction as compared to native orcontrol levels. In some instances, the reduction can be a 100%reduction. It is understood that treatment does not necessarily refer toa cure or complete ablation of the disease, condition, or symptoms ofthe disease or condition. As used herein, references to decreasing,reducing, or inhibiting include a change of 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater as compared to a control level. Such termscan include, but do not necessarily include, complete elimination.

The subject on which the method is performed has been diagnosed withhemophilia A. The subject is mammalian, including humans; non-humanprimates, such as apes and monkeys; cattle; horses; sheep; rats; dogs;cats; mice; pigs; and goats. In some embodiments, the subject is ahuman, a dog, a horse, a sheep, a cow, or a cat. The subject may be maleor female. The subject may be a juvenile subject or an adult subject. Asa recessive X-linked disorder, a male subject will carry an X chromosomethat has a mutation in the Factor VIII gene. A female subject that hashemophilia A will either have a mutated Factor VIII allele on both Xchromosomes or will have a mutant Factor VIII allele on one X chromosomeand have an inactive Factor VIII allele on the other X chromosome. Insome instances, the subject may be a female carrier of hemophilia A thathas a mutant Factor VIII allele on one X chromosome and a normal FactorVIII gene on the other X chromosome. Subjects may be diagnosed viaprenatal genetic testing, particularly in instances where there is afamily history of hemophilia. The DNA from biological samples obtainedfrom amniocentesis, chorionic villi sampling, or cell-free fetal DNApresent in the maternal peripheral blood may be analyzed forabnormalities on the X chromosome or mutations in the Factor VIII gene.Alternatively, subjects may be diagnosed after birth by assessing theability of the subject's blood to clot properly. For example, screeningtests include activated partial thromboplastin time (APTT) test,prothrombin time (PT) test, and fibrinogen test. Diagnosis of hemophiliaA (type and severity) can also be performed with antigen-based teststhat assess the amount of Factor VIII protein in the subject's blood. Insome instances, the subject may have received therapy with infusedFactor VIII protein. Where the subject has received such therapy, insome instances the subject may have developed an immune response toFactor VIII protein (developed inhibitory antibodies that impair theeffectiveness of the therapy). In some embodiments, the subject hasreceived prior treatment with exogenous Factor VIII and has developed aninhibitory immune response that diminishes the effectiveness of theexogenous Factor VIII treatment.

MSC, referred to in the field as mesenchymal stem cells, mesenchymalstromal cells, and, when isolated from bone marrow, also marrow stromalprogenitors (MSP), are multipotent stromal cells that can differentiateinto a variety of cell types, including: osteoblasts, chondrocytes,myocytes, and adipocytes. MSC do not have the capacity to reconstitutean entire organ. The term encompasses multipotent cells derived fromother non-marrow tissues, such as placenta, umbilical cord blood,adipose tissue, or the dental pulp of deciduous baby teeth. MSC areheterogeneous and different subsets of MSC may have differentcapabilities. Different methods of isolation will result in differentpopulations of MSC. Such different populations may express differentprotein markers. MSC subpopulations with different marker expressionprofiles have been found to have different capabilities. See, forexample, Thierry, D., et al., Stro-1 Positive and Stro-1 Negative HumanMesenchymal Stem Cells Express Different Levels of Immunosuppression,Blood 104(11): 4964 (2004). The extent to which a MSC populationisolated using one method and having a particular marker profile willshare properties with a MSC population isolated using a different methodand having a different marker profile has not been determined.

In some instances, the MSC used in the method are bone marrow-derivedMSC—that is, the MSC are isolated from bone marrow. Specifically, thebone marrow-derived MSC are isolated from bone marrow obtained from thesubject (autologous MSC). In some instances, the bone marrow-derived MSCused in the method express Stro-1, CD146, or both Stro-1 and CD146. Flowcytometry methods may be used to isolate MSC expressing these markerssuch as described, for example, in Sanada C., et al., Mesenchymal stemcells contribute to endogenous FVIII:c production. J Cell Physiol. 2013;228(5):1010-1016 and Chamberlain J. L., et al., Efficient generation ofhuman hepatocytes by the intrahepatic delivery of clonal humanmesenchymal stem cells in fetal sheep. Hepatology. 2007;46(6):1935-1945. Isolating MSC based on Stro-1 and/or CD146 results in adistinct cell population from that isolated using the traditionalapproach in which bulk unpurified bone marrow or Ficoll-purified bonemarrow mononuclear cells are plated directly into plastic cell cultureplates or flasks to which the adherent MSC population binds.

In some instances, the MSC used in the method are MSC isolated from abirth tissue or birth fluid. Specifically, the MSC may be isolated fromamniotic fluid, placental tissue, chorionic villi, or umbilical cordtissue. In some instances, the MSC used in the method express c-kit.Methods of isolating such cells are described in U.S. Pat. Nos.7,968,336 and 8,021,876, which are incorporated herein by reference intheir entirety. In some instances, the MSC express at least one ofc-kit, CD34, CD90, or CD133. In some instances, the MSC express c-kitand at least one of CD34, CD90, or CD133. In some instances, the MSC areisolated based on expression of c-kit.

For juvenile patients for whom prenatal biological samples areavailable, the MSC may be isolated from such samples (such as amnioticfluid, placental, cord tissue). Such samples may be available where thesubject is diagnosed with hemophilia prior to birth. In some instances,appropriate biological samples may be obtained at the time of thesubject's birth (such as amniotic fluid, placental, cord tissue). Foradult patients, or juvenile patients for which prenatal biologicalsamples are not available, the MSC used in the method may be bone marrowderived mesenchymal stem/stromal cells (MSC), also referred to as bonemarrow stromal cells.

The MSC used in the method are modified to express high levels of FactorVIII. In some instances, the MSC may be modified to also express highlevels of von Willebrand factor (vWF).

In some instances, an exogenous gene sequence encoding one or both ofthese proteins may be introduced into the MSC via one or more vectors.In some instances, the MSC may be modified to express high levels ofFactor VIII protein via introduction into the MSC of a vector comprisinga Factor VIII gene sequence operatively linked to a constitutivelyactive promoter. In some instances, the MSC may be modified to expresshigh levels of vWF protein via introduction into the MSC of a vectorcomprising a vWF gene sequence operatively linked to a constitutivelyactive promoter. In some instances, the MSC may be modified to expresshigh levels of Factor VIII protein and vWF protein via introduction intothe MSC of a vector comprising a Factor VIII gene sequence operativelylinked to a constitutively active promoter and a vector encoding a vWFgene sequence operatively linked to a constitutively active promoter. Insome instances, the Factor VIII gene sequence and the vWF gene sequencemay be operatively linked to the same constitutively active promoter.Alternatively, the Factor VIII gene sequence and the vWF gene sequencemay be operatively linked to different constitutively active promoters.

Exemplary vectors include, for example, plasmids and viral vectors(including but not limited to adenoviral, adeno-associated viral (AAV),or retroviruses such as lentiviruses. In preferred embodiments, thevector is a viral vector. In some instances, the vector may be a vectorthat integrates into the genome of transduced cells. For example, thevector may be a lentivirus vector. In preferred embodiments, the vectoris a lentivirus vector. In some instances, the lentivirus vectorcontains a 3′-modified long terminal repeat (LTR), resulting in aself-inactivating (SIN) lentivector. A lentivirus vector may integrateinto the genome of dividing or non-dividing cells. The lentivirus genomein the form of RNA is reverse-transcribed to DNA when the virus entersthe cell, and is then inserted into the genome by the viral integraseenzyme. The lentivirus vector, now called a provirus, remains in thegenome and is passed on to the progeny of the cell when it divides. Inanother example, the vector may be an adeno-associated virus (AAV)vector, which, in contrast to wild-type AAV, only rarely integrates intothe genome of the cells it transduces. In one example, the vector may bean adenoviral vector. An adenoviral vector does not integrate into thegenome. In another instance, the vector may be a murine retrovirusvector. In another example, the vector may be a foamy virus vector,which may have a larger capacity for inserts than lentiviral vectors. Inanother example, the vector may be Sendai virus vector.

The exogenous gene sequences are operatively linked to one or morepromoter sequences within the vector. The term “promoter sequence” or“promoter element” refers to a nucleotide sequence that assists withcontrolling expression of a coding sequence. Generally, promoterelements are located 5′ of the translation start site of a gene.However, in certain embodiments, a promoter element may be locatedwithin an intron sequence, or 3′ of the coding sequence. In someembodiments, a promoter useful for a gene therapy vector is derived fromthe native gene of the target protein (e.g., a Factor VIII promoter). Insome embodiments, the promoter is a constitutive promoter, which drivessubstantially constant expression of protein from the exogenous genesequence. Non-limiting examples of well-characterized promoter elementsinclude the cytomegalovirus immediate-early promoter (CMV), the β-actinpromoter, the methyl CpG binding protein 2 (MeCP2) promoter, the simianvirus 40 early (SV40) promoter, human Ubiquitin C promoter (UBC), humanelongation factor 1α promoter (EF1α), the phosphoglycerate kinase 1promoter (PGK), or the CMV immediate early enhancer/chicken beta actin(CAG) promoter. The vector will generally also contain one or more of apromoter regulatory region (e.g., one conferring constitutiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

In some instances, the Factor VIII transgene is operably linked to apromoter. A number of promoters can be used in the practice of theinvention. The promoters can be selected based on desired outcome. Thenucleic acids can be combined with constitutive, inducible,tissue-preferred, or other promoters for expression in the organism ofinterest. See, for example, promoters set forth as SEQ ID NOs: 1-6 asdescribed in Brown et al. (2018) Target-Cell-Directed BioengineeringApproaches for Gene Therapy of Hemophilia A. Mol. Ther. Methods Clin.Dev., 2018. 9:57-69, which is herein incorporated by reference in itsentirety for all purposes.

Where the MSC are modified to express high levels of Factor VIII viatransduction with an exogenous Factor VIII gene sequence, the exogenousFactor VIII gene sequence may be human Factor VIII gene sequence,porcine Factor VIII gene sequence, or a hybrid transgene comprisingportions of human Factor VIII gene sequence and portions of porcineFactor VIII gene sequence. In some instances, the gene sequencecomprises all or a portion of the human Factor cDNA as set forth inGenBank Accession No. 192448441 as updated Jul. 17, 2017, wherein saidportion would encode a function portion of the human Factor VIIIprotein. In some instances, the gene sequence comprises a sequence thatis at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to all or a portion ofthe human Factor cDNA as set forth in GenBank Accession No. 192448441 asupdated Jul. 17, 2017, wherein said portion would encode a functionalportion of the human Factor VIII protein. In some instances, the genesequence may encode all or a functional portion of the human Factor VIIIprotein as set forth in GenBank Accession No. 192448441 as updated Jul.17, 2017, reflecting the protein transcribed from transcript variant 1of the Factor VIII gene. This protein is approximately 300 kDa andcontains a series of homology-defined domains designatedA1-A2-B-ap-A3-C₁-C₂. In some instances, the exogenous Factor VIII genesequence is modified relative to wild-type protein sequence to result inincreased protein expression, increased protein stability, reducedimmunogenicity, or a combination of one or more thereof.

In some instances, the sequence of one or more of the Factor VIIIprotein domains may be deleted. In one example, the B domain of FactorVIII is deleted. The B domain of Factor VIII has no known function andcan be deleted without loss of coagulant activity. Deletion of theB-domain has been shown to increase factor VIII protein production inheterologous systems (Toole et al. (1986) Proc. Natl. Acad. Sci. U.S.A.83:5939-5942). In addition, wildtype porcine Factor VIII protein havingthe B-domain deleted may have 10-100-fold higher expression andsecretion than the human Factor FVIII gene sequence, both in vitro andin vivo. (See, for example, Dooriss, K. L., et al., Comparison of factorVIII transgenes bioengineered for improved expression in gene therapy ofhemophilia A. Hum Gene Ther. 20:465-478 (2009). A B-domain deleted formof human Factor VIII protein (Lind et al. (1995) Eur. J. Biochem.232:19-27) has been approved for clinical use.

In some instances, the exogenous Factor VIII gene sequence may includeprotein modifications to reduce immunogenicity of the protein therebyreducing the risk of an immune response due to therapy. For example,alanine substitutions may be included as described in Healey, J. F., etal., The comparative immunogenicity of human and porcine factor VIII inhaemophilia A mice. Thromb Haemost. 102:35-41 (2009) and Lubin, I. M.,et al., Analysis of the human factor VIII A2 inhibitor epitope byalanine scanning mutagenesis. J Biol Chem. 272:30191-30195 (1997), whichare incorporated by reference herein in their entirety.

In some instances, one or more of the human Factor VIII protein domainsequences may be substituted with the sequence of the correspondingporcine Factor VIII protein domain sequences. For example, one or moreporcine Factor VIII domains may be substituted for one or more humanFactor VIII domains. For example, inclusion of the porcine Factor VIIIdomains A1 and ap-A3 may increase expression of the expressed FactorVIII protein. See, for example, Doering, C. B., et al., Identificationof porcine coagulation factor VIII domains responsible for high levelexpression via enhanced secretion. J Biol Chem. 279:6546-6552 (2004). Insome embodiments, the exogenous Factor VIII gene sequence may comprisethe human Factor VIII A2 and C2 domains and the porcine Factor VIII A1,A3, and C1 domains.

In some instances, the exogenous Factor VIII gene sequence may comprisea modified Factor VIII sequence comprising a B domain-deleted (BDD)Factor VIII transgene having the sequence of the human A2 and C2 domainsand the porcine A1, A3, and C1 domains, and also include three alaninesubstitutions in the A2 domain to reduce immunogenicity, as described inLubin, I. M., et al., Analysis of the human factor VIII A2 inhibitorepitope by alanine scanning mutagenesis. J Biol Chem. 1997;272(48):30191-5. This modified Factor VIII protein is referred to as theET3 transgene in this disclosure, including in the Examples below. Insome instances, the ET3 transgene is expressed at a comparable level tothat of wild-type porcine Factor VIII protein while having 91% identityto the amino acid sequence of wild-type human Factor VIII protein. Inone example, the exogenous Factor VIII gene sequence may comprise ahuman/porcine Factor VIII transgene as described in Doering, C. B., etal., Directed engineering of a high-expression chimeric transgene as astrategy for gene therapy of hemophilia A, Mol. Ther. 17(7):1145-1154(2009), which is incorporated herein by reference in its entirety.

In some instances, the Factor VIII transgene sequence may comprise oneof the modified Factor VIII sequences described in Brown et al. (2018)Target-Cell-Directed Bioengineering Approaches for Gene Therapy ofHemophilia A. Mol. Ther. Methods Clin. Dev., 2018. 9:57-69, which isincorporated herein by reference in its entirety for all purposes.Factor VIII polypeptides, including tissue-specific codon optimizedvariants, are described therein. Modified Factor VIII transgenesequences used in the methods described herein can be any one of SEQ IDNOs: 7-16 (as described in Brown et al.). For example, Factor VIIItransgene sequences that can be used in the methods described hereininclude a B-domain deleted (BDD) human Factor VIII polypeptide (HSQ) asset forth in SEQ ID NO: 15, a BDD chimeric human/porcine Factor VIIIpolypeptide (ET3) as set forth in SEQ ID NO: 11, or an ancestral FactorVIII polypeptide (An53) as set forth in SEQ ID NO: 7.

In some instances, the exogenous Factor VIII gene sequence may bemodified for expression in a particular organ or tissue type. Forexample, the gene sequence may be optimized for expression in myeloidtissue. In some embodiments, the Factor VIII transgene may comprisemyeloid codon optimized ET3 (mcoET3) as set forth in SEQ ID NO: 12 ormyeloid codon optimized HSQ (mcoHSQ) as set forth in SEQ ID NO: 16.Alternatively, the Factor VIII transgene may be optimized for expressionin liver tissue. In some embodiments, the Factor VIII transgene maycomprise liver codon optimized ET3 (lcoET3) as set forth in SEQ ID NO:10; liver codon optimized An53 as set forth in SEQ ID NO: 8; or livercodon optimized (lcoHSQ) as set forth in SEQ ID NO: 14.

In some instances, the exogenous Factor VIII gene sequence may compriseone of the modified Factor VIII sequences described in U.S. Pat. No.7,635,763, which is incorporated herein by reference in its entirety forall purposes. Regions of the porcine Factor VIII polypeptide thatcomprises the A1 and ap-A3 regions, and variants and fragments thereof,are described therein that impart high-level expression to both theporcine and human Factor VIII polypeptide. The exogenous Factor VIIIgene sequence encoded by the viral vector of the provided methods may bethe polynucleotides set forth in any one of SEQ ID NOs: 19, 21, 23, 25,or 27 (SEQ ID NOs: 15, 17, 19, 13, or 21 as described in U.S. Pat. No.7,635,763). The modified Factor VIII protein expressed at high levels inthe modified MSC may comprise the amino acid sequences set forth in anyone of SEQ ID NOs: 18, 20, 22, 24, or 26 (SEQ ID NOs: 14, 16, 18, 12, or20 as described in U.S. Pat. No. 7,635,763). Such sequences aresummarized in Table 1 below. In some instances, these sequences may beused to construct an exogenous Factor VIII gene sequence encoding amodified factor VIII polypeptide that results in a high level ofexpression of the encoded modified Factor VIII protein.

TABLE 1 Exemplary Modified Factor VIII Proteins Modified Factor VIIIProtein SEQ ID NO. Description HP44/OL aa: SEQ ID NO: 18A1_(P)-A2_(P)-ap_(P)-A3_(P)-C1_(H)-C2_(H) nt: SEQ ID NO: 19 porcine A1,A2, ap-A3 domains, porcine- derived linker sequence S F A Q N S R P P SA S A P K P P V L R R H Q R (SEQ ID NO: 30), and human C1 and C2 domainsHP46/SQ aa: SEQ ID NO: 20 A1_(P)-A2_(H)-ap_(H)-A3_(H)-C1_(H)-C2_(H) nt:SEQ ID NO: 21 porcine A1 domain, human A2, ap-A3, C1 and C2 domains, andhuman S F S Q N P P V L K R H Q R linker sequence HP47/OL aa: SEQ ID NO:22 A1_(P)-A2_(H)-ap_(p)-A3_(P)-C1_(H)-C2_(H) nt: SEQ ID NO: 23 porcineA1, ap-A3 domains, porcine- derived linker sequence S F A Q N S R P P SA S A P K P P V L R R H Q R (SEQ ID NO: 31), and human A2, C1 and C2domains B-domain deleted aa: SEQ ID NO: 24 Human Factor VIII proteinsequence nt: SEQ ID NO: 25 minus B-domain HP63/OL aa: SEQ ID NO: 26porcine A1 domain and a partially nt: SEQ ID NO: 27 humanized ap-A3domain that comprises porcine amino acids from about 1690 to about 1804and from about 1819 to about 2019

As discussed above, in some instances, the MSC are also modified toexpress high levels of vWF protein via introduction into the MSC of avector. In some embodiments coding sequences for vWF can be any one ofSEQ ID NOs: 28 or 29. In some instances, the vWF gene sequence in thevector may encode all or a functional portion of the human vWF proteinset forth in GenBank Accession No. 1023301060 as updated Aug. 21, 2017.However, in some instances, the vWF gene sequence may include one ormore modifications to the wild-type vWF gene sequence to increaseprotein expression, increase protein stability, reduce immunogenicity,or a combination of one or more thereof, of the vWF protein. Forexample, the full cDNA sequence of the vWF gene may be too large to bepackaged efficiently in certain vectors, such as, for example, alentiviral vector. Thus, in some instances, one or more exons of the vWFgene may be deleted while still retaining biological function of theexpressed protein. In some instances, exons 24-46 of the vWF gene may bedeleted as described in U.S. Patent Application Publication No.2010/0183556. In some instances, the vWF gene sequence may becodon-optimized for efficient expression in the MSC. In some instances,the exogenous vWF gene sequence may modified for expression in aparticular organ or tissue type. For example, the gene sequence may beoptimized for expression in the liver. Thus, in some instances, the vWFgene sequence may comprise at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 90%, 89% 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identity tothe corresponding wild-type vWF gene sequence and comprise modificationsto improve expression. In some instances, the vWF gene sequencecomprises the truncated human vWF sequence set forth below in thisdisclosure or a sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 90%, 89% 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identicalthereto while retaining biological activity of the expressed protein. Insome instances, the vWF gene sequence comprises the truncated sheep vWFsequence set forth below in this disclosure or a sequence at least 99%,98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%, 87%, 86%, 85%, 84%,83%, 82%, 81%, or 80% identical thereto while retaining biologicalactivity of the expressed protein.

In some instances, gene-editing may be performed on the MSC to insert,delete, or replace the genomic sequence of one or both of the endogenousgenes using engineered nucleases (molecular scissors). Gene-editingnucleases belong to one of three known categories: zinc-finger nucleases(ZFN), transcription activator-like effector nucleases (TALEN), andclustered regularly interspaced short palindromic repeats (CRISPR) andtheir associated proteins (Cas) tools. All operate on the sameprinciple; they are all capable of inducing a double-strand break at adefined genomic sequence that is subsequently corrected by endogenousDNA repair mechanisms. Double-strand breaks can be repaired throughhomology-driven repair (HDR), in the presence of donor homologous DNAsequences, resulting in gene-editing events.

In some instances, the MSC may be modified to express high levels of theFactor VIII protein via gene-editing of an endogenous Factor VIII genesequence of the MSC, wherein the gene-editing introduces one or moremodifications to an endogenous Factor VIII gene sequence that increaseprotein expression, increase protein stability, reduce immunogenicity,or a combination of one or more thereof, of the Factor VIII protein. Insome instances, the MSC are modified to express high levels of anexogenous FVIII protein via genome-editing, wherein the gene-editingintroduces an exogenous FVIII gene, under the control of a constitutivepromoter, into a “safe harbor” region within the genome, such as theAAVS1 site. In some instances, the MSC are modified to express highlevels of the vWF protein via gene-editing of an endogenous vWF genesequence of the MSC, wherein the gene editing introduces one or moremodifications to the endogenous vWF gene sequence that increase proteinexpression, increase protein stability, reduce immunogenicity, or acombination of one or more thereof, of the vWF protein. In someinstances, the MSC are modified to express high levels of an exogenousvWF protein via genome-editing, wherein the gene-editing introduces anexogenous vWF gene, under the control of a constitutive promoter, into a“safe harbor” within the genome, such as the AAVS1 site. Exemplary “safeharbor” regions are described in Cerbini, T., et al., Transfection,selection, and colony-picking of human induced pluripotent stem cellsTALEN-targeted with a GFP gene into the AAVS1 safe harbor. J Vis Exp.2015 Feb. 1; (96):52504 and Hong, S. G., et al., Rhesus iPSC Safe HarborGene-Editing Platform for Stable Expression of Transgenes inDifferentiated Cells of All Germ Layers. Mol Ther. 2017; 25(1):44-53.

In some instances, the endogenous Factor VIII gene sequence may bemodified by gene-editing to have the type of modifications describedabove for embodiments where an exogenous Factor VIII gene sequence isintroduced via transduction. The discussion of the various modificationsdescribed above is thus also applicable to embodiments where theendogenous Factor VIII gene sequence is modified. For example, in someinstances, the sequence of one or more protein domains of the endogenousFactor VIII gene sequence may be deleted. In some instances, the Bdomain of Factor VIII is deleted. In some instances, the endogenousFactor VIII gene sequence may be modified to reduce immunogenicity ofthe protein thereby reducing the risk of an immune response due totherapy. For example, alanine substitutions may be introduced asdescribed in Healey, J. F., et al., The comparative immunogenicity ofhuman and porcine factor VIII in haemophilia A mice. Thromb Haemost.102:35-41 (2009) and Lubin, I. M., et al., Analysis of the human factorVIII A2 inhibitor epitope by alanine scanning mutagenesis. J Biol Chem.272:30191-30195 (1997), which are incorporated by reference herein intheir entirety.

In some instances, the endogenous Factor VIII gene sequence may bemodified to substitute one or more of the Factor VIII protein domainsequences with the sequence of the corresponding Factor VIII proteindomain sequences from another species. For example, for human subjects,the endogenous Factor VIII gene sequence may be modified to substituteone or more of the human Factor VIII protein domain sequences with thesequence of the corresponding porcine Factor VIII protein domainsequences. For example, substitution with the porcine Factor VIIIdomains A1 and ap-A3 may increase expression of the expressed FactorVIII protein. See, for example, Doering, C. B., et al., Identificationof porcine coagulation factor VIII domains responsible for high levelexpression via enhanced secretion. J Biol Chem. 279:6546-6552 (2004). Insome embodiments, the endogenous Factor VIII gene sequence may bemodified to comprise the porcine Factor VIII A1, A3, and C1 domains,while retaining the human Factor VIII A2 and C2 domains.

In some instances, the endogenous Factor VIII gene sequence may bemodified to include a B domain deletion, the porcine A1, A3, and C1domains, and also include three alanine substitutions in the A2 domainto reduce immunogenicity, as described above for the exogenous FactorVIII gene sequence embodiments. In one example, the endogenous FactorVIII gene sequence may be modified to have the sequence of ahuman/porcine Factor VIII transgene as described in Doering, C. B., etal., Directed engineering of a high-expression chimeric transgene as astrategy for gene therapy of hemophilia A, Mol. Ther. 17(7):1145-1154(2009), which is incorporated herein by reference in its entirety. Insome instances, the modified endogenous Factor VIII gene sequenceresults in expression of a modified Factor VIII protein at a levelcomparable to that of wild-type porcine Factor VIII protein while having91% identity to the amino acid sequence of wild-type human Factor VIIIprotein.

In some instances, the endogenous Factor VIII gene sequence may bemodified to comprise one of the modified Factor VIII sequences describedin U.S. Pat. No. 7,635,763, which is incorporated herein by reference inits entirety for all purposes. In some instances, the endogenous FactorVIII gene sequence may comprise the polynucleotides set forth in any oneof SEQ ID NOs: 19, 21, 23, 25, or 27 (SEQ ID NOs: 15, 17, 19, 13, or 21as described in U.S. Pat. No. 7,635,763). The modified Factor VIIIprotein expressed at high levels in the modified MSC may comprise theamino acid sequences set forth in any one of SEQ ID NOs: 18, 20, 22, 24,or 26 (SEQ ID NOs: 14, 16, 18, 12, or 20 as described in U.S. Pat. No.7,635,763). Such sequences are summarized in Table 1 above.

As discussed above, in some instances, the MSC are also modified toexpress high levels of vWF protein via gene-editing. In some instances,the vWF gene sequence may include one or more modifications to thewild-type vWF gene sequence to increase protein expression, increaseprotein stability, reduce immunogenicity, or a combination of one ormore thereof, of the vWF protein. For example, in some instances, one ormore exons of the vWF gene may be deleted while still retainingbiological function of the expressed protein. In some instances, exons24-46 of the vWF gene may be deleted as described in U.S. PatentApplication Publication No. 2010/0183556. In some instances, the vWFgene sequence may be codon-optimized for efficient expression in theMSC. In some instances, the exogenous vWF gene sequence may modified forexpression in a particular organ or tissue type. For example, the genesequence may be optimized for expression in the liver. Thus, in someinstances, the vWF gene sequence may be modified to comprise at least99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%, 87%, 86%, 85%,84%, 83%, 82%, 81%, or 80% identity to the corresponding wild-type vWFgene sequence and comprise modifications to improve expression. In someinstances, the vWF gene sequence may be modified to comprise thetruncated human vWF sequence set forth below in this disclosure or asequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%,87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical thereto whileretaining biological activity of the expressed protein. In someinstances, the vWF gene sequence may be modified to comprise thetruncated sheep vWF sequence set forth below in this disclosure or asequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%,87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical thereto whileretaining biological activity of the expressed protein.

Where the MSC are modified to express high levels of both Factor VIIIand vWF, the same method of modification may be used to achieve highexpression of both proteins or different methods could be used for eachprotein. For example, in some instances, the MSC may be modified toexpress high levels of both Factor VIII and vWF protein via introductionof exogenous gene sequences for both proteins. In another example, theMSC may be modified to express high levels of both Factor VIII and vWFprotein via gene-editing of the endogenous gene sequences of bothproteins. In some instances, the MSC may be modified to express highlevels of Factor VIII via transduction of an exogenous Factor VIII genesequence and modified to express high levels of vWF via gene-editing ofthe endogenous vWF gene sequences. In other instances, the MSC may bemodified to express high levels of vWF via transduction of an exogenousvWF gene sequence and modified to express high levels of Factor VIII viagene-editing of the endogenous Factor VIII gene sequences.

A “high level of expression” means that the production/expression of themodified Factor VIII protein or vWF protein is at an increased level ascompared to the expression level of the corresponding native Factor VIIIprotein or vWF protein expressed under the same conditions. An increasein protein expression levels (considered a high level of expression)comprises at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 20-fold or greater expression of the modified Factor VIIIprotein or vWF protein compared to the expression levels of thecorresponding Factor VIII protein or vWF protein. Alternatively,“high-level expression” can comprise an increase in protein expressionlevels of at least 1-25 fold, 1-5 fold, 5-10 fold, 10-15 fold, 15-20fold, 20-25 fold or greater expression levels of the modified FactorVIII protein or vWF protein when compared to the corresponding FactorVIII protein or vWF protein. Methods for assaying protein expressionlevels are routine in the art. By “corresponding” Factor VIII protein orvWF protein is intended a Factor VIII protein or vWF protein thatcomprises an equivalent amino acid sequence. In one example, expressionof a modified human Factor VIII protein comprising the A1-A2-ap-A3-C1-C2domains is compared to a human Factor VIII protein containingcorresponding domains A1-A2-ap-A3-C1-C2. In another example, for afragment of a modified human Factor VIII protein containing domainsA1-A2-ap-A3, expression is compared to a fragment of human Factor VIIIprotein having the corresponding domains A1-A2-ap-A3. Alternatively, incertain instances, expression of a modified Factor VIII protein or vWFprotein may be compared to the full-length corresponding proteins. Inone example, for a fragment of a modified human Factor VIII proteincontaining domains A1-A2-ap-A3, expression is compared to human FactorVIII protein having the A1-A2-ap-A3-C1-C2 domains.

The modified MSC are cultured in vitro to generate an expanded modifiedMSC population. The expanded modified MSC population provides sufficientnumbers of modified MSC for therapeutic use. Culture conditions may beselected based on the type of MSC used in the method. For example, MSCisolated from placental tissue may be grown in culture medium optimizedfor placental cells. In another example, MSC isolated from amnion tissuemay be grown in culture medium optimized for amniotic cells. In anotherexample, MSC isolated from umbilical cord or bone marrow may be grown inculture medium optimized for MSC cells. The modified cells may be grownon plastic culture dishes for at least 2, 3, 4, 5, or 6 passages togenerate the expanded modified MSC population. In some instances, all ora portion of the expanded modified MSC population may be cryopreserved.

Following culturing of the modified MSC to generate expanded modifiedMSC population, modified MSC from expanded modified MSC population areinjected into the subject. The injection may be made at least one ofintraperitoneal injection, intravenous injection, or intra-articularinjection. Each injection comprises about 10⁵ to about 10⁹ MSC from theexpanded modified MSC population per kilogram weight of the subject. Forexample, the injection may comprise 10⁵ MSC, 10⁶ MSC, 10⁷ MSC, 10⁸ MSC,or 10⁹ MSC. The number of cells injected into the subject is based onthe amount of protein expressed per cell. This metric is determinedempirically for the expanded modified MSC population. In some instances,this metric may be generally predictable based on the nature of themodified MSC (for example, method of modification, Factor VIII genesequence, vWF gene sequence, vector and vector components).

In some instances, modified MSC are injected into the subject once,twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or10 times. In some instances, modified MSC are injected into the subjectat least once, at least twice, at least 3 times, at least 4 times, atleast 5 times, at least 6 times, at least 7 times, at least 8 times, atleast 9 times, or at least 10 times. For example, the modified MSC mayinjected as multiple injections on the same day. In some instances, themodified MSC may be injected into the subject on multiple days. In someinstances, the subject is injected with modified MSC on a first day andthen the subject may be monitored over a period of time (days or weeks)to determine if there is sufficient protein expression to provide thedesired therapeutic benefit. In some instances, the amount of proteinexpression in the subject's blood of Factor VIII protein, vWF protein,or both, may be monitored. In some instances, the efficiency of thesubject's blood to clot may be assessed using routine blood clottingtests known in the art. In some instances, the subject's symptomsrelating to joint pain and/or inflammation may be assessed. Wheremonitoring indicates that the amount of expression of Factor VIIIprotein alone, or the amount of expression of Factor VIII protein andvWF protein, is insufficient, the subject's disease symptoms are notalleviated, or both, the subject may be injected with modified MSC on asecond day. Again, the subject may be monitored over a period of time todetermine if there is sufficient protein expression to provide thedesired therapeutic benefit. These steps may be repeated for a fourth,fifth, sixth, seventh, eighth, ninth, or tenth injection as needed toachieve the desired therapeutic benefit of alleviating the subject'sdisease symptoms.

In some instances, the use of MSC as cellular vehicles to deliver aFactor VIII gene sequence, a vWF gene sequence, or both, to a subject(as opposed to administration of vector directly) may overcomelimitations/risks observed to-date in AAV-based clinical trials forhemophilia: 1) the possibility of off-target transduction of troublingcell types, such as germline cells; 2) the inability to treat patientswith pre-existing antibodies to the serotype of AAV being employed as avector; and 3) the transient hepatotoxicity induced by the AAV capsid,that triggers subsequent immune/inflammatory destruction of many of thetransduced cells. Although early studies in vitro and in normal andhemophilia A mice, have used unselected stromal cells (isolated basedsolely upon plastic adherence) as cellular vehicles for deliveringexogenous Factor VIII, no attempts have yet been made to usephenotypically-defined MSC/pericytes to deliver FVIII in vivo in anypreclinical model of hemophilia A.

In some instances, the use of MSC as cellular vehicles to deliver atherapeutic gene is also an improvement over the use of hematopoieticstem cells (HSC), as have been used in most cell-based gene therapytrials. The use of MSC eliminates the possibility of insertionalleukemogenesis, which is the most serious adverse event seen to-date inclinical gene therapy trials. A successful outcome of the proposedstudies targeting hemophilia A thus promises to open the door to safecorrection of a variety of congenital disorders using MSC to deliver thetherapeutic gene.

EXAMPLES Example 1. Animal Model

Applicant re-established a line of sheep that emulates the genetics,inhibitor formation, and clinical symptoms of the severe form of humanhemophilia A (HA), including the development of frequent, spontaneoushematomas and crippling hemarthroses, making them unique among the HAmodels. See Porada, C. D., et al., Clinical and molecularcharacterization of a re-established line of sheep exhibiting hemophiliaA. J Thromb Haemost, 2010. 8(2): 276-285. Using unique antibodiesdeveloped to various regions of the ovine FVIII protein, it wasdetermined that these sheep do not produce any FVIII antigen (FIG. 1),as demonstrated by the complete lack of any staining within the liver oftwo of the HA sheep, which is in marked contrast to the widespreadbright staining that is seen in the liver from a normal/healthy sheep.As such, they should be an excellent model of severe, cross-reactingmaterial (CRM)-negative hemophilia A patients. Additionally, sheep areclose in size to humans, their immune system is quite similar to that ofhumans, and their long lifespan allows long-term efficacy and safety tobe addressed.

Example 2. Preliminary Pilot Study—Allogeneic Cells

A pilot study on 2 pediatric subjects from the HA sheep model describedin Example 1. See Porada, et al., Phenotypic correction of hemophilia Ain sheep by postnatal intraperitoneal transplantation ofFVIII-expressing MSC. Exp Hematol, 2011. 39(12):1124-1135. During thefirst 3-5 months of life, both animals had received frequent, on-demandinfusions of human FVIII (hFVIII) for multiple hematomas and chronic,progressive, debilitating hemarthroses of the leg joints which hadresulted in severe defects in posture and gait, rendering them nearlyimmobile. Thus, for these subjects, FVIII was presented in the contextof “danger signals”, which is known to trigger a robust host immuneresponse to FVIII and other proteins.

Haploidentical MSC from the ram that had sired the two HA lambs wereused for the therapy. The MSC were modified to introduce viatransduction a B domain-deleted, wild-type porcine FVIII cDNA asdescribed in Porada et al., Phenotypic correction of hemophilia A insheep by postnatal intraperitoneal transplantation of FVIII-expressingMSC. Exp Hematol. 2011; 39(12):1124-1135. MSC were simultaneouslytransduced with 2 lentivectors; one encoded eGFP for in vivo tracking ofdonor cells, and the second encoded an expression/secretion optimizedporcine FVIII (pFVIII) transgene previously shown to beexpressed/secreted from human cells at 10-100 times higher levels thanhFVIII or sheep (ovine) FVIII (oFVIII). See Gangadharan et al.,High-level expression of porcine factor VIII from genetically modifiedbone marrow-derived stem cells. Blood, 2006. 107(10):3859-64; Doering etal., Directed Engineering of a High-expression Chimeric Transgene as aStrategy for Gene Therapy of Hemophilia A. Mol Ther, 2009.17(7):1145-54; Doering et al., Identification of porcine coagulationfactor VIII domains responsible for high level expression via enhancedsecretion. J Biol Chem, 2004. 279(8):6546-52; Dooriss et al., Comparisonof Factor VIII Transgenes Bioengineered for Improved Expression in GeneTherapy of Hemophilia A. Hum Gene Ther, 2009. 20(5):465-78; Ide, L. M.,et al., Hematopoietic stem-cell gene therapy of hemophilia Aincorporating a porcine factor VIII transgene and nonmyeloablativeconditioning regimens. Blood, 2007. 110(8):2855-63; and Johnston et al.,Generation of an optimized lentiviral vector encoding a high-expressionfactor VIII transgene for gene therapy of hemophilia A. Gene Ther, 2013.20(6):607-15.

FVIII/GFP-expressing MSC were then expanded and transplanted by IPinjection (30×10⁶), in the absence of any preconditioning, into thefirst lamb. Following transplant, this animal's clinical pictureimproved dramatically, and he enjoyed an event-free clinical course,devoid of spontaneous bleeds, obviating the need for hFVIII infusions.Even more remarkably, the animal's existing hemarthroses resolved, hisjoints recovered fully, and he regained normal posture and gait,resuming a normal activity level. To the inventors' knowledge, thisrepresents the first report of phenotypic correction of severe HA in alarge animal model following transplantation of cells modified toexpress FVIII, and is the first time that reversal of chronicdebilitating hemarthroses has been achieved in any setting.

Based on this remarkable clinical improvement, the modified MSC weretransplanted into the second animal using an identical procedure, but ahigher cell dose (125×10⁶). In similarity to the first animal,hemarthroses present in this second animal at the time of transplantresolved, he resumed normal activity shortly after transplantation, andbecame factor-independent.

Interestingly, despite the marked phenotypic improvement in both theseanimals, no circulating FVIII activity was detectable following thetransplant, most likely due to the presence of high-titer inhibitors inthese animals. These findings are remarkable, since despite the hightiters of antibodies/inhibitors present in these animals, thetransplanted allogeneic (haploidentical) MSC persisted and were noteliminated by the recipient's immune system, and the therapeutic effectof the treatment was maintained, i.e., the animals' symptoms ofspontaneous joint bleeds, hematomas, and bleeding upon needle stick allimproved.

Example 3. Mechanistic Study—Autologous Cells

Twenty female HA carriers will be artificially inseminated (AI) vialaparoscopy, as done in Example 2, with the support of the NorthCarolina State Theriogenology/Ruminant Medicine team. At 50-70 days ofgestation (term: 150 days), amniotic fluid will be collected, and fetalcells from the amniotic fluid will be isolated, cultured, and expanded,using standard methods in our lab. Given the severe phenotype of theseanimals, we will perform a PCR-based RFLP (see Porada, C. D., et al., JThromb Haemost, 2010. 8(2):276-85) to identify affected fetuses,allowing us to plan for their subsequent delivery. Followingamniocentesis, the animals will be allowed to complete term. When thesheep have nearly completed gestation, the pregnant ewes carryingaffected fetuses will be placed under close observation, and ewes willeither be induced into labor using intramuscular dexamethasone fornatural delivery, or the lambs will be delivered by Caesarian section,with clinical veterinarians assisting in either case. Both approacheshave been used previously with success.

Affected lambs will be treated immediately with recombinant full-lengthor B-domain deleted ovine FVIII (oFVIII) produced as described in Zakas,P. M., et al., Development and characterization of recombinant ovinecoagulation factor VIII. PLoS One, 2012. 7(11):e49481. Although we havefound that oFVIII is not a very high-expressing FVIII variant whencompared to FVIII from other species, oFVIII protein for transfusion andan oFVIII transgene in the vectors are being used because the consensusin the hemophilia field is that the use of “same-species” FVIII isessential in preclinical gene therapy-based studies to accurately modelthe potential immune response in the clinical arena.

While human cells may be needed to perform definitive clinical studies,human cells are not appropriate for these mechanistic studies becauseusing human cells would not allow us to address the critical issue ofwhether the use of autologous cells results in higher levels oflong-term engraftment than we achieved in our pilot study withallogeneic cells, and whether the use of autologous cells may reduce theincidence of inhibitor formation. For this reason, sheep MSC will beused throughout this study.

It is our goal to treat HA with this MSC-based delivery system duringthe first 18 months of postnatal human life, since this is the time bywhich most HA patients would be diagnosed. Sheep develop much fasterthan humans, and are weaned at 60-90 days of age. We thus know thiscorresponds to the first 12-18 months for a human, so we will test theMSC-based treatment during the first 2-3 months of life in the sheep.

Starting at birth, HA lambs will be treated prophylactically 2-3 timesper week with recombinant oFVIII. At 4-5 weeks of age, we will collectbone marrow (under oFVIII coverage), and isolate MSC from each affectedlamb, as we have done previously. These methods enable us tosuccessfully establish primary sheep MSC that are phenotypically andfunctionally similar to their human counterparts; these sheep MSC aredevoid of hematopoietic cells (they lack CD11b, CD34, and CD45), butthey express the MSC markers CD146 and CD90. See FIG. 2. Anti-ovineantibodies to other antigens routinely used to identify MSC, such asCD44, CD105, and CD73, are not commercially available.Immunofluorescence microscopy demonstrated expression of vimentin andα-smooth muscle actin, known MSC cytoskeleton proteins, and we verifiedthe ability of these sheep MSC were able to differentiate intoadipocytes (by Oil-red-o staining) and osteocytes (by calcium depositionand alkaline phosphatase). See FIG. 2.

We will then subject the isolated MSC to 2-3 rounds of transduction witheither the EF1α-[oFVIII] lentivirus vector (FIG. 3) alone, orsimultaneously with the EF1α-[oFVIII] lentivirus vector and theCAG-[vWF] lentivirus vector (FIG. 4). See De Meyer, S. F., et al.,Phenotypic correction of von Willebrand disease type 3 blood-derivedendothelial cells with lentiviral vectors expressing von Willebrandfactor. Blood, 2006. 107(12): 4728-36. The EF1α-[oFVIII] lentivirusvector contains a B-domain deleted oFVIII gene having the polynucleotidesequence set forth in SEQ ID NO: 33. The CAG-[vWF] lentiviral vectorcontains a truncated vWF having the polynucleotide sequence set forth inSEQ ID NO: 29. From the pilot study described in Example 2, we know that2-3 rounds of simultaneous exposure to two different lentivirus vectorsresults in transduction of 90-95% of sheep MSC with both vectors. Thelentivirus vectors for these studies both contain the 3′-modified LTR toproduce SIN lentivirus vectors, and the constitutive EF1α promoter willdrive FVIII expression. Due to packaging constraints, it is not possibleto utilize the EF1α promoter in the vWF lentivirus vector. Prior studieshave established the ability of vWF to be packaged within a lentivirusvector and expressed at high levels. See De Meyer, S. F., et al., 2006(above). However, if any difficulties arise packaging vWF-encodinglentivirus vectors, we will utilize truncated vWF cassettes as describedin this disclosure, or switch to foamy virus vectors, as they possess amuch larger packaging capacity.

We are including a group in which autologous MSC are transduced withvectors encoding both oFVIII and vWF (ovine vWF; GI:426227037) for tworeasons: 1) binding to vWF stabilizes FVIII and prolongs its half-lifeand, thus, delivery of MSC secreting FVIII complexed with vWF mayproduce a more pronounced therapeutic effect; and 2) vWF may reduce theimmunogenicity of exogenously administered FVIII by preventing both itsuptake and presentation by dendritic cells, and its recognition byimmune effector cells. We predict that delivering the two proteins inthe same vector will result in the release of FVIII:vWF as a complexfrom the transduced MSC. We will confirm co-localization/complexformation of vector-derived oFVIII and vWF in transduced MSC populationsby confocal microscopy prior to performing the proposed transplants.Although MSC do not endogenously produce any vWF, we will add a 6-Histag (SEQ ID NO:32) to the vWF transgene, making it readilydistinguishable from any trace endogenous vWF for these in vitrostudies.

Following transduction, the FVIII-expressing MSC will be expanded untilthe animals have reached 2-3 months of age, at which point the MSC betransplanted autologously into their respective donor HA lamb, via IPinjection under ultrasound guidance, with no preconditioning (as in ourpilot study [23]), using a dose of 5-10×10⁶ cells/kg. An aliquot of eachcell type will be reserved to determine vector copy number by qPCR andto perform integration site analysis by LM-PCR. Methods described inPorada, C. D., et al., Phenotypic correction of hemophilia A in sheep bypostnatal intraperitoneal transplantation of FVIII-expressing MSC. ExpHematol, 2011. 39(12): 1124-1135 (qPCR) and Russo-Carbolante, E. M., etal., Integration pattern of HIV-1 based lentiviral vector carryingrecombinant coagulation factor VIII in Sk-Hep and 293T cells. BiotechnolLett, 2011. 33(1):23-31 and Tellez, J., et al., High Incidence of VectorIntegration Near Cancer Related Genes within Primitive HematopoieticStem Cells (HSC) After Fetal Gene Transfer with γ-Retroviral Vectors.Molecular Therapy, 2010. 18(Suppl. 1): p. S331 (LM-PCR). Twoexperimental groups will be included: 1) autologous MSC transduced withthe EF1α-[oFVIII] lentivector (n=2-3 HA lambs); and 2) autologous MSCtransduced with both the EF1α-[oFVIII] and CAG-vWF lentivectors (n=2-3HA lambs).

Following transplantation, prophylactic oFVIII infusions will bediscontinued, and any benefit as a result of this MSC-based approachshould be readily apparent, given the severe, life-threatening phenotypeof these animals. The sheep will be continually monitored for bleeds,and platelet-deficient plasma will be collected monthly until at least1.5 years of age for coagulation assays, to quantify the plasma levelsof oFVIII by chromogenic assay and/or ELISA. The formation/presence ofinhibitors will be assessed at each time point by performing theNijmegen modification of the Bethesda assay (as described in Verbruggen,B., et al., The Nijmegen modification of the Bethesda assay for factorVIII:C inhibitors: improved specificity and reliability. Thromb Haemost,1995. 73(2):247-51) and a commercially available kit(Technoclone/DiaPharma Group, Inc.) on an aliquot of plasma collectedfrom the animals. Once we have obtained these values, we will comparethe HA lambs that received MSC transduced with the lentivector encodingoFVIII alone to those that received MSC transduced with both the oFVIIIand vWF lentivectors, and compare each of these to the historic valuesfrom untransplanted HA sheep, and to a reference panel of normalunaffected males. These studies will allow us to: 1) establish whatlevels of vector-encoded oFVIII are expressed as a result of thispostnatal approach; 2) determine the duration of the therapeutic effectof this approach; 3) assess whether using autologous MSC and alentivector that lacks eGFP avoids the inhibitors seen in the pilotstudy of Example 2; and 4) establish whether including vWF improves thetherapeutic effect of this MSC-based treatment and/or reduces theincidence/titer of inhibitor formation.

At 1.5 years of age (or sooner, if we see that FVIII levels aredropping), the HA lambs will be euthanized, and all major organs will beharvested. All tissues will be fixed in 4% paraformaldehyde, processedthrough a sucrose gradient, embedded and frozen in OCT, and sectioned at5 μm. 8-10 slides/tissue will be stained with an antibody specific tooFVIII and analyzed/quantitated by confocal microscopy for the presenceof engrafted MSC to precisely determine the levels and localization(parenchymal vs. perivascular) of engrafted cells that are expressingFVIII, and are therefore providing therapeutic benefit. We will alsocollect plasma from each of these recipients at the time of euthanasia,and quantitate the circulating levels of vector-derived oFVIII in thesesheep using an ELISA specific for oFVIII, correlating levels andpatterns of engraftment with circulating FVIII levels. Based on ease ofaccess to the circulation, we hypothesize that maximal plasma FVIIIlevels will be obtained when MSC lodge in perivascular regions of theengrafted tissues.

While confocal analysis should provide us with a fairly accurateestimate of the levels of oFVIII+MSC within each tissue, the slidesselected for quantitation may or may not be representative of theengraftment levels within the organ as a whole. Therefore, we will alsouse an ELISA to precisely quantitate the amount of oFVIII within tissuehomogenates. A standard curve will be created with known numbers ofoFVIII+MSC, thereby establishing how much oFVIII is present on aper-cell basis. Protein extracts will then be prepared from the tissuesfrom each animal and analyzed using this ELISA, comparing the tissuevalues to that of the standard curve, to precisely quantitate the numberof MSC that have engrafted within each tissue and are expressing oFVIII.We will then compare the levels of donor MSC in each tissue with theresultant plasma FVIII levels to determine in which tissues engraftmentproduces the highest circulating levels of FVIII.

Example 4. Study Assessing Treatment in Subjects with Pre-ExistingInhibitors

The findings of the study described in Example 3 will be used todetermine the ability of this autologous cell-based approach to mediateclinical/phenotypic improvement in recipients with pre-existinginhibitors as a result of on-demand FVIII treatment.

Three to four HA lambs will be treated on-demand with oFVIII beginningat birth. We expect, based on the pilot study of Example 2, thattreating on-demand with FVIII products will result in the formation ofinhibitors in almost all HA sheep by 4-5 months of age. We will collectbone marrow and isolate MSC at 4-5 weeks of age, and transduce thesecells with either the EF1α-[oFVIII] lentivector alone, or with both theEF1α-[oFVIII] and CAG-vWF lentivectors (depending which approach yieldsthe best outcome in the preceding studies), and expand the transducedcells to obtain adequate numbers for transplant.

Beginning at birth, we will draw blood from these animals every otherweek to obtain plasma and perform the Nijmegen modification of theBethesda assay (as above) (Technoclone/DiaPharma Group, Inc.) to assessthe development of inhibitors. Once inhibitors have developed, we willtransplant the transduced autologous MSC into each animal byultrasound-guided IP injection, as in Exp. Set #1.1, at a dose of5-10×10⁶ cells/kg.

Following transplantation, we will analyze the animals as detailed inExample 3, continually monitoring them for bleeds, and collectingplatelet-deficient plasma monthly until at least 1 year of age forcoagulation assays, to quantify the plasma levels of oFVIII bychromogenic assay and/or ELISA, and to quantitate the levels ofinhibitors present, to ascertain whether the MSC-based treatment impactsupon the levels of the pre-existing inhibitors. We will then compare theresults obtained with these HA lambs with pre-existing inhibitors tothose in Example 3 in which the HA lambs lacked inhibitors at the timeof MSC infusion.

Example 5. First Line Therapy Study

We hypothesize that previously untreated patients (PUPS) represent theideal group to initially target with this MSC-based treatment, becausetheir immune systems are completely naïve to exogenous FVIII, and willbe exposed to it for the first time when it is released by thetransplanted MSC; we anticipate this will reduce/eliminate the risk ofinhibitor formation in this population.

In families with no prior history, HA is normally diagnosed during thefirst 18 months of life, after the child exhibits abnormalbruising/bleeding after a minor trauma. In families with a history ofHA, diagnosis can be made at birth, or even prior to birth [148-158].Regardless of when diagnosis is made, however, it would not be possibleto collect bone marrow MSC from these patients without treating withfactor to prevent hemorrhage during the procedure. This same issueexists with the HA sheep, since they present with a severe phenotype andspontaneous bleeding from birth. However, in similarity to patients witha family history of HA, the affected sheep can be diagnosed in utero byamniocentesis (as can be done in human patients), making it possible tocollect autologous cells from the amniotic fluid part way throughgestation, transduce these cells, expand them, and have them ready totransplant as the first-line therapy at birth, or shortly thereafter. Werecently found that MSC-like cells present within the amniotic fluid,“AF-MSC”, are readily transduced with lentivirus vectors, theyendogenously produce low levels of FVIII (FIG. 5) and vWF (FIG. 6), andthey express very high levels of FVIII following lentivectortransduction (FIG. 7). These findings suggest that amniotic fluid canreplace marrow as a source of autologous MSC for delivering a FVIIItransgene, enabling us to test the MSC-based treatment's efficacy asfirst-line therapy in PUPs.

To test the efficacy of the MSC-based treatment in PUPs, HA carrier eweswill be bred or artificially inseminated as detailed in Example 3. At50-60 days of gestation (term: 150 days), amniotic fluid will becollected, AF-MSC isolated, and a PCR-based RFLP performed to detect theHA mutation, as detailed in Example 3. AF-MSC from the affected fetuseswill then be subjected to 2-3 rounds of transduction with theEF1α-[oFVIII] comprising the polynucleotide sequence set forth in SEQ IDNO: 33 (FIG. 3) and expanded. At near term, labor will be induced in theewes with affected lambs, or a C-section performed to deliver the lambs.Immediately after birth, the HA lamb “PUPs” (n=3-4) will be injected IPwith the transduced autologous AF-MSC at a dose of 5-10×10⁶ cells/kg, asdescribed for the BM-MSC in Example 3.

Following transplantation, we will analyze the animals as detailed inExample 3 and 4, continually monitoring them for bleeds, and collectingplatelet-deficient plasma monthly until at least 1 year of age for: 1)coagulation assays; 2) to quantify the plasma levels of oFVIII bychromogenic assay and/or ELISA; and 3) to perform the Nijmegen modifiedBethesda assay to assess the development of inhibitors. We will thencompare the results obtained by using this MSC-based treatment as afirst-line therapy in these HA lamb “PUPS” to the results obtained inthe HA lambs treated prophylactically (Example 3) and to those treatedon-demand (Example 4) prior to MSC infusion.

Example 6. Study to Assess Induction of Immune Tolerance to Factor VIII

We hypothesize that the continued delivery of FVIII to the circulationby the lentivector-modified MSC can serve as a much-needed novel methodof inducing immune tolerance to FVIII.

The aim of this study is to test the ability of this MSC-based approachas a novel method of inducing immune tolerance through the continueddelivery of FVIII to the circulation by the genetically-modified MSC. Toaccomplish this objective, 2-3 HA lambs (more will be added if initialdata are not clear-cut) will be treated on-demand with oFVIII, beginningat birth, as we know that treating on-demand with FVIII products resultsin the formation of inhibitors in almost all HA sheep by 4-5 months ofage. As in Example 4, we will isolate BM-MSC at 4-5 weeks of age, andtransduce these cells with both the EF1α-[oFVIII] and CAG-vWFlentivectors, as clinical data indicate that the inclusion/presence ofvWF may facilitate ITI [123, 159, 160]. The transduced cells will thenbe expanded to obtain adequate numbers for subsequent transplant.

Beginning at birth, we will draw blood from these animals every otherweek to obtain plasma and perform the Nijmegen modified Bethesda assay(as above) to assess inhibitor induction. Once inhibitors havedeveloped, we will transplant transduced autologous MSC, at a dose of10′ cells/kg, into the peritoneal cavity of each animal, as in Example3. This procedure will be repeated each 4-5 days until we observe a dropin inhibitor titer (as detailed below); a maximum of 10 infusions willbe given initially.

Following transplantation, we will analyze the animals as in Example 3,continually monitoring for bleeds (as the repeated MSC-based treatmentshould produce clinical/phenotypic improvement), and collecting plateletdeficient plasma bi-weekly for ≥3 months, to perform coagulation assays,to quantify the oFVIII plasma levels by chromogenic assay and/or ELISA,and to quantitate the levels of inhibitors present, to ascertain whetherthe repeated infusion of MSC expressing high levels of FVIII can breakthe existing inhibitors and induce tolerance to FVIII. To furtherconfirm that this cell-based ITI has overcome the existing inhibitors,we will assess the restoration of normal FVIII pharmacokinetics, usingwell-established methodology (plasma FVIII recovery≥66% of expected anda ≥6 h half-life, determined following a 72-hour FVIII-exposure-freeperiod).

Example 7. Comparison of Recombinant Factor VIII Infusion Therapy withMSC-Based Tolerance Induction

One of the only clinical options for HA patients who develop inhibitorsis immune tolerance induction (ITI), which involves the long-termadministration of high doses of FVIII protein. ITI is extremelyexpensive, is only effective in a percentage of patients withinhibitors, and the mechanism for its success is unknown. To-date, nopreclinical HA model has been used to study and/or optimize ITI. Giventhe high incidence of inhibitor formation in the HA sheep and their lackof any cross-reactive material, they represent an excellent model inwhich to investigate ITI. We propose to perform a head-to-headcomparison of traditional ITI, using repeated high-dose recombinantoFVIII to the MSC-based ITI protocol developed/tested in Example 6.

To achieve these goals, 2-3 HA lambs will be treated on-demand withoFVIII, beginning at birth, as described in Example 6, until inhibitorsdevelop. We will then commence a clinically employed, protein-based ITIregimen, infusing the inhibitor animals with a dose of 100 IU/kg/day for3 months (as described in Oldenburg, J., et al., Primary and rescueimmune tolerance induction in children and adults: a multicentreinternational study with a VWF-containing plasma-derived FVIIIconcentrate. Haemophilia, 2014. 20(1):83-91). During the course of thisITI protocol, we will analyze the animals as detailed in Example 6,collecting platelet-deficient plasma weekly, to: 1) perform coagulationassays; 2) quantify the plasma levels of oFVIII by chromogenic assayand/or ELISA; and 3) quantitate the levels of inhibitors present, toassess the ability of the protein-based ITI to break existinginhibitors, and define the kinetics with which this happens. To furtherconfirm that ITI has overcome existing inhibitors, we will assess therestoration of normal FVIII pharmacokinetics, as detailed above. Thesuccess rate and kinetics of tolerance induction with the protein-basedITI will be compared to those of the cell-based protocol in Example 6,to determine whether the cell-based method is a viable alternative tothe time consuming and expensive protein-based method that representsthe current state-of-the-art in clinical ITI.

Example 8. Transduction Efficiency with Different Vectors

The transduction efficiency, FVIII production, and FVIII secretion fromhuman PLC following transduction at an identical multiplicity ofinfection (MOI) of 7.5 with an identical lentiviral vector (LV) encodingone of the following four different FVIII transgenes: (1) abioengineered human-porcine hybrid FVIII (ET3) having the polynucleotidesequence set forth in SEQ ID NO: 11; (2) a liver codon-optimized ET3(lcoET3) having the polynucleotide sequence set forth in SEQ ID NO: 10;(3) a liver codon-optimized human FVIII (lcoHSQ) having thepolynucleotide sequence set forth in SEQ ID NO: 14; and (4) amyeloid-codon optimized ET3 (mcoET3) having the polynucleotide sequenceset forth in SEQ ID NO: 12 were compared. Brown et al. (2018) Mol. Ther.Methods Clin. Dev. 9:57-69, demonstrated that vectors encoding FVIII,when codon-optimized to the target cells, or tissue, result in adramatically increase FVIII expression of functional FVIII. Followingtransduction, PLCs were analyzed by flow cytometry and confocalmicroscopy to measure transduction efficiency and FVIII production.Conditioned media of PLCs were assayed by aPTT to quantitate FVIIIactivity. Analysis of the culture supernatants by aPTT demonstratedFVIII activity was readily detectable in supernatants of all transducedcells lines. It also revealed marked differences in the secretion offunctional FVIII following transduction with each of these vectors.Specifically, PLCs transduced with mcoET3 (SEQ ID NO: 12), ET3 (SEQ IDNO: 11), lcoET3 (SEQ ID NO: 10), and lcoHSQ (SEQ ID NO: 14) LV secreted25±9, 19±8, 11±2, and 1±0.1 IU of FVIII/24 h/10⁶ cells, respectively(FIG. 10). PLC population doubling time was not affected by transductionwith any of the vectors; nor were phenotype or expression of signalingmolecules involved in innate immunity. Importantly, at passage 3following transduction with any of the 4 lentiviral vectors, PLCscontinued to produce and secrete FVIII at similar levels to thoseobserved shortly after transduction, demonstrating stable vectorintegration and durability/longevity of FVIII expression. The relativelevels of FVIII expression by PLCs following transduction with eachlentiviral vector were also assessed by immunofluorescence microscopywith an antibody specific to a region of FVIII that is conserved in all4 FVIII transgenes. These analyses confirmed the results of the aPTTanalyses on the supernatants from these cells, with mcoET3-PLCexhibiting the brightest/highest intensity staining for FVIII, followedby PLC transduced with ET3 (SEQ ID NO: 11), then those transduced withlcoET3 (SEQ ID NO: 10) and with lcoHSQ (SEQ ID NO: 14) (data not shown).

The gene transfer efficiency of these gene-modified cells was assessedby determining the final proviral/vector copy number (VCN) using acommercially available qPCR-based kit (Lenti-X Provirus QuantitationKit, Takara Bio USA, Inc., Mountain View, Calif.). To ensure that onlyintegrated copies were detected by the assay, qPCR for VCN was performedin PLCs that had been passaged at least three times after transduction.After transducing the cells at the same MOI (7.5) with each lentiviralvector, the VCNs for mcoET3-PLC, lcoHSQ-PLC, lcoET3-PLC, and ET3-PLCwere all around 1.

Example 9. Optimizing Factor VIII Expression in Placental Cells

The aim of this study was to investigate the suitability of placentalcells (PLC) as cellular delivery vehicles for FVIII. The expression ofphenotypical markers was determined in three different master cellsbanks (101, 103, and 104) of placental cells (PLCs), each of which wasderived from a different human donor by the Regenerative MedicineClinical Core (RMCC) at WFIRM following GMP-compliant standard operatingprocedures (SOPs) established by the RMCC for PLC. Expression of CD29,CD44, CD73, CD90, CD105, HLA-ABC, HLA-E, CD31, CD34, CD35, CD144, HLA-G,HLA-DR/DP/DQ, and ABO blood group were determined using flow cytometricanalysis. These markers were selected to confirm that the PLC isolatedpossessed a phenotype characteristic of MSC from other tissues (CD29,CD44, CD73, CD90, CD105), to assess their potential for stimulating animmune response upon transplantation (HLA-ABC, HLA-E, CD35, CD144,HLA-G, HLA-DR/DP/DQ, and ABO blood group), and to discern whether theyexpressed markers indicative of endothelial properties (CD31, CD34). Nostatistically significant differences (p<0.05) were found in expressionof phenotypic markers between PLCs derived from three different mastercell banks (101, 103, and 104). PLCs from each of the master cell banksexpressed CD29, CD44, CD73, CD90, CD 105, HLA-ABC, and HLA-E (FIG. 8A);had negligible amounts (<1%) of CD31, CD34, CD35, CD144, HLA-G, andHLA-DR/DP/DQ (data not shown); and were devoid of ABO blood group (datanot shown). Collectively, these findings support the conclusion that PLCare an MSC-like population and that they should exhibit minimalimmunogenicity upon transplantation.

PLCs derived from each of the master cell banks [MCBs] (101, 103, and104) were assessed for their ability to express FVIII proteinconstitutively. Immunofluorescence microscopy with a primary antibodyspecific to hFVIIIc and a fluorochrome-conjugated secondary antibody andflow cytometric analysis with fluorochrome-conjugated antibodies to wereused to determine the levels of constitutively expressed FVIII proteinand define the phenptype of the PLCs, respectively. As shown in FIG. 8A,these cells expressed markers characteristic of MSC from bone marrow andother tissues. All three MCBs endogenously expressed detectable amountsof FVIII by immunofluorescence microscopy, and MCB 103 expressed thehighest levels, as indicated by the brightest fluorescence intensity(data not shown). The fold increase of FVIII expression over isotypecontrol for PLCs derived from each of the MCBs is presented as relativemean fluorescence intensity (MFI) as shown in FIG. 8B.

The activated partial thromboplastin time (aPTT or PTT) assay is afunctional measure of the intrinsic and common pathways of thecoagulation cascade (i.e. it characterizes blood coagulation). The aPTTassay was used to quantitate levels of functional FVIII secreted byPLCs. PLCs were plated at the same density and cultured for 24 hours inPhenol Red-free alpha-MEM AmnioMax Complete Medium (ThermoFisherScientific, Raleigh, N.C.). Supernatants were collected and the numberof cells present counted, and then the levels of secreted functionalFVIII were measured by the Clinical Hematology Laboratory at Wake ForestBaptist Health using a commercial aPTT assay. Levels of FVIII werenormalized by adjusting to account for the number of cells present atthe time of supernatant collection, and expressing FVIII activity on aper cell basis. The data from this analysis is shown in FIG. 8C

FVIII mRNA levels in the PLCs derived from three different master cellbanks (101, 103, and 104) were evaluated by qPCR using primers specificto human FVIII. Relative expression of endogenous mRNA for FVIII wascalculated by comparing the threshold cycle (CT) value for FVIII withthe CT of each master cell bank's respective internal reference gene,GAPDH. The relative expression of endogenous FVIII mRNA was 0.01±0.0005,0.075±0.007, and 0.011±0.0002, for PLCs 101, 103, and 104, respectively.

Example 10. Suitability of PLCs as a Transgenic FVIII ProductionPlatform

PLC 101, 103, and 104 were transduced at the same MOI (7.5) using alentiviral vector-(LV) encoding mcoET3 (mcoET3-PLC) as described inExample 4 above. Vector copy number (VCN) was determined, as describedin detail above. The VCN was found to be similar between the threedifferent PLC MCBs (0.71-0.75). After transduction, the relative levelsof expression of FVIII by the 3 MCB PLCs were assessed byimmunofluorescence microscopy after staining with a primary antibodyspecific to hFVIIIc and a fluorochrome-conjugated secondary antibody.All 3 MCBs expressed high levels of FVIII after transduction with themcoET3 lentiviral vector, but MCB 103 exhibited the highest levels ofFVIII protein, as evidence by the brightest/highest fluorescenceintensity (data not shown). The secretion of FVIII was determined usingaPTT performed on 24-hour culture supernatants harvested from PLCs thatwere plated at the same density and normalized for the number of cellspresent at the time of the supernatant collection, as described indetail in the preceding paragraphs (FIG. 9A). Levels of FVIII in theculture supernatant increased significantly (p<0.05) in PLCs derivedfrom all 3 MCBs (101, 103, and 104) when compared with respectivenon-transduced PLC counterparts (FIG. 9B). No significant differenceswere found between the different transduced cells.

The effect of transduction of PLCs with LV encoding mcoET3 on phenotypeor molecules involved in immunity was assessed. Expression of CD29,CD44, CD73, CD90, CD 105, CD58, CD112, CD155, CD47, HLA-ABC, HLA-E,HLA-G, and HLA-DR/DP/DQ, were determined by flow cytometric analysis, asdescribed above. No statistically significant differences (p<0.05) werefound between transduced and non-transduced cells (FIGS. 8A-8C).Additionally, PLC population doubling time was not affected bytransduction (data not shown). Both non-transduced and transduced PLCsexpressed CD47, a molecule involved in immune evasion (FIG. 8B).Transduced cells did not significantly upregulate the expression ofHLA-DR/DP/DQ (FIG. 8C).

To further examine whether transduction of the PLC with the mcoET3lentiviral vector had the potential to alter the immunogenicity of thesecells, we examined the levels of expression of various Toll-likeReceptors (TLRs) on the PLC prior to and following transduction, asthese molecules play a key role in innate immunity, and theirupregulation could potentially trigger an immune response to thetransduced cells upon transplantation. To address this possibility, theeffect of PLC transduction with the mcoET3 lentiviral vector on TLR-3,TLR-4, TLR-7, TLR-8, and TLR-9 expression was assessed. TLR expressionon transduced (t) and non-transduced (n) PLCs (101, 103, and 104) wasdetermined using flow cytometric analysis. No significant differences inexpression of TLR molecules was detected in the PLC populations. Asshown in FIG. 11A, there was no difference in the levels of expressionof any of these TLRs in transduced vs. non-transduced PLCs, confirmingthat transduction of these 3 MCB PLCs with the mcoET3 lentiviral vectordid not lead to upregulation of any of these immune-stimulatingmolecules.

In order to evaluate the demands of PLC transduction with mcoET3 andincreased Factor VIII expression on the secretory and endoplasmicreticulum pathways, expression of stress molecules MICA/B, ULBP-1,ULBP-2, and ULBP-3 was determined in transduced (t) and non-transduced(n) PLCs (101, 103, and 104). Flow cytometric analysis demonstrated thatno significant expression or alteration/upregulation of MICA/B or ULBP-1were found before or after transduction with mcoET3 lentiviral vector(FIG. 11B).

The production of interferon-gamma (IFN-γ) by mcoET3 transduced andnon-transduced PLCs was measured using a high-sensitivity ELISA (assayrange: 0.16-10.0 pg/mL). PLCs were cultured for 24 hours in AmnioMaxComplete Medium (ThermoFisher). Supernatants were collected and IFN-γproduction was determined. No IFN-γ was detected in any of the culturesupernatants of mcoET3-transduced or non-transduced PLCs (data notshown).

All features of the described systems are applicable to the describedmethods mutatis mutandis, and vice versa.

All patents, patent publications, patent applications, journal articles,books, technical references, and the like discussed in the instantdisclosure are incorporated herein by reference in their entirety forall purposes.

It is to be understood that the figures and descriptions of thedisclosure have been simplified to illustrate elements that are relevantfor a clear understanding of the disclosure. It should be appreciatedthat the figures are presented for illustrative purposes and not asconstruction drawings. Omitted details and modifications or alternativeembodiments are within the purview of persons of ordinary skill in theart.

It can be appreciated that, in certain aspects of the disclosure, asingle component may be replaced by multiple components, and multiplecomponents may be replaced by a single component, to provide an elementor structure or to perform a given function or functions. Except wheresuch substitution would not be operative to practice certainembodiments, such substitution is considered within the scope of thedisclosure.

The examples presented herein are intended to illustrate potential andspecific implementations of the invention. It can be appreciated thatthe examples are intended primarily for purposes of illustration forthose skilled in the art. There may be variations to these diagrams orthe operations described herein without departing from the spirit of theinvention. For instance, in certain cases, method steps or operationsmay be performed or executed in differing order, or operations may beadded, deleted or modified.

Different arrangements of the components depicted in the drawings ordescribed above, as well as components and steps not shown or describedare possible. Similarly, some features and sub-combinations are usefuland may be employed without reference to other features andsub-combinations. Aspects and embodiments of the invention have beendescribed for illustrative and not restrictive purposes, and alternativeembodiments will become apparent to readers of this patent. Accordingly,the present invention is not limited to the embodiments described aboveor depicted in the drawings, and various embodiments and modificationscan be made without departing from the scope of the claims below.

While exemplary embodiments have been described in some detail, by wayof example and for clarity of understanding, those of skill in the artwill recognize that a variety of modification, adaptations, and changesmay be employed. Hence, the scope of the present invention should belimited solely by the claims.

1. A method of treating a subject diagnosed with hemophilia A,comprising: (a) modifying mesenchymal stem/stromal cells (MSC) toexpress high levels of Factor VIII protein or high levels of both FactorVIII protein and von Willebrand factor (vWF) protein thereby generatingmodified MSC, the MSC comprising bone-marrow MSC isolated from thesubject; (b) generating an expanded modified MSC population by in vitroculturing the modified MSC; and (c) injecting MSC from the expandedmodified MSC population into the subject.
 2. The method of claim 1,wherein isolating the MSC comprises isolating cells that express atleast one of Stro-1 or CD146.
 3. The method of claim 1, wherein thesubject has received prior treatment with exogenous Factor VIII and hasdeveloped an inhibitory immune response that diminishes theeffectiveness of the exogenous Factor VIII treatment.
 4. A method oftreating a subject prenatally diagnosed as having hemophilia A,comprising: (a) modifying mesenchymal stem/stromal cells (MSC) toexpress high levels of Factor VIII protein or high levels of both FactorVIII protein and von Willebrand factor (vWF) protein thereby generatingmodified MSC, the MSC comprising MSC isolated from at least one ofamniotic fluid, placental tissue, or umbilical cord tissue obtained atthe time of the subject's birth or prenatally from the subject's mother;(b) generating an expanded modified MSC population by in vitro culturingthe modified MSC; and (c) injecting MSC from the expanded modified MSCpopulation into the subject.
 5. The method of claim 4, wherein isolatingthe MSC comprises isolating cells that express c-kit.
 6. The method ofclaim 4, wherein the isolated MSC express c-kit, CD34, CD90, and CD133.7. (canceled)
 8. (canceled)
 9. The method of claim 1, wherein themodified MSC comprise one or both of a Factor VIII gene sequence or avWF gene sequence comprising one or more modifications that increaseprotein expression, protein stability, or both, of the Factor VIIIprotein or vWF protein.
 10. (canceled)
 11. (canceled)
 12. The methodclaim 1, wherein the MSC are modified by introducing into the MSC aviral vector comprising a Factor VIII gene sequence operatively linkedto a constitutively active promoter, a viral vector comprising a vWFgene sequence operatively linked to a constitutively active promoter, ora viral vector comprising a Factor VIII gene sequence operatively linkedto a constitutively active promoter and a vWF gene sequence operativelylinked to a constitutively active promoter.
 13. The method claim 1,wherein the MSC are modified via gene editing, wherein the gene editingintroduces one or more modifications to one or both of an endogenousFactor VIII gene sequence or an endogenous vWF gene sequence thatincrease protein expression, protein stability, or both.
 14. (canceled)15. (canceled)
 16. The method of claim 12, wherein the Factor VIII genesequence and the vWF gene sequence are operatively linked to the sameconstitutively active promoter.
 17. (canceled)
 18. The method of claim1, wherein the MSC from the expanded modified MSC population areinjected into the subject via at least one of intraperitoneal injection,intravenous injection, or intra-articular injection.
 19. The method ofclaim 1, wherein the expanded modified MSC population are injected intothe subject at least once, at least twice, or at least three times. 20.The method of claim 1, wherein the expanded modified MSC population areinjected into the subject in an amount of about 10⁷ to about 10⁹ MSC perkilogram weight of the subject.
 21. The method of claim 4, wherein themodified MSC comprise one or both of a Factor VIII gene sequence or avWF gene sequence comprising one or more modifications that increaseprotein expression, protein stability, or both, of the Factor VIIIprotein or vWF protein.
 22. The method claim 4, wherein the MSC aremodified by introducing into the MSC a viral vector comprising a FactorVIII gene sequence operatively linked to a constitutively activepromoter, a viral vector comprising a vWF gene sequence operativelylinked to a constitutively active promoter, or a viral vector comprisinga Factor VIII gene sequence operatively linked to a constitutivelyactive promoter and a vWF gene sequence operatively linked to aconstitutively active promoter.
 23. The method of claim 22, wherein theFactor VIII gene sequence and the vWF gene sequence are operativelylinked to the same constitutively active promoter.
 24. The method claim4, wherein the MSC are modified via gene editing, wherein the geneediting introduces one or more modifications to one or both of anendogenous Factor VIII gene sequence or an endogenous vWF gene sequencethat increase protein expression, protein stability, or both.
 25. Themethod claim 4, wherein the MSC from the expanded modified MSCpopulation are injected into the subject via at least one ofintraperitoneal injection, intravenous injection, or intra-articularinjection.
 26. The method claim 4, wherein the expanded modified MSCpopulation are injected into the subject at least once, at least twice,or at least three times.
 27. The method claim 4, wherein the expandedmodified MSC population are injected into the subject in an amount ofabout 10⁷ to about 10⁹ MSC per kilogram weight of the subject.